Antibodies Directed Against Lymphocyte Activation Gene 3 (LAG-3)

ABSTRACT

The invention relates to an isolated immunoglobulin heavy chain polypeptide and an isolated immunoglobulin light chain polypeptide that bind to a protein encoded by the Lymphocyte Activation Gene-3 (LAG-3). The invention provides a LAG-3-binding agent that comprises the aforementioned immunoglobulin heavy chain polypeptide and immunoglobulin light chain polypeptide. The invention also provides related vectors, compositions, and methods of using, the LAG-3-binding, agent to treat a disorder or disease that is responsive to LAG-3 inhibition, such as cancer or an infectious disease.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 182,600 Byte ASCII (Text) file named“723163_ST25.TXT,” created on Feb. 2, 2016.

BACKGROUND OF THE INVENTION

Lymphocyte Activation Gene-3 (LAG-3), which is also known as CD223, is amember of the immunoglobulin supergene family and is structurally andgenetically related to CD4. LAG-3 is expressed on T-cells, B cells,natural killer (NK) cells and plasmacytoid dendritic cells (ADCs). LikeCD4, LAG-3 has been demonstrated to interact with MHC Class 11 molecules(Baixeras et al., J. Exp. Med., 176: 327-337 (1992)), but binds at adistinct site (Huard et al., Proc. Natl. Acad. Sci. USA, 94(11):5744-5749 (1997)). In, particular, for example, a LAG-3 immunoglobulinfusion protein (sLAG-3Ig) directly and specifically binds via LAG-3 toMHC class II on the cell surface (Huard et al., Eur. J. Immunol.,26:1180-1186 (1996)).

LAG-3 is upregulated following T-cell activation, and modulates T-cellfunction as well as T-cell homeostasis (Sierra et al., Expert Opin.Ther. Targets, 15(1):91-101 (2011)). The LAG-3/MHC class 11 interactionmay play a role in down-regulating antigen-dependent stimulation of CD4+T lymphocytes, as demonstrated in in vitro studies of antigen-specificT-cell responses in which the addition of anti-LAG-3 antibodies led toincreased T-cell proliferation, higher expression of activation antigenssuch as CD25, and higher concentrations of cytokines such asinterferon-gamma and interleukin-4 (Huard et al., Eur. J. Immunol., 24:3216-3221 (1994)). CD4+CD25+ regulatory T-cells (Treg) also have beenshown to express LAG-3 upon activation and antibodies to LAG-3 inhibitsuppression by induced Treg cells, both in vitro and in vivo, suggestingthat LAG-3 contributes to the suppressor activity of Treg cells (Huanget al. Immunity, 21: 503-513 (2004)). Furthermore, LAG-3 has been shownto negatively regulate T-cell homeostasis by regulatory T-cells in bothT-cell-dependent and independent mechanisms (Workman, C. J. and Vignali,D. A., J. Immunol., 174: 688-695 (2005)).

Subsets of conventional T-cells that are anergic or display impairedfunctions express LAG-3, and LAG-3+ T-cells are enriched at tumor sitesand during chronic viral infections. However, while LAG-3 knockout micehave been shown to mount normal virus-specific CD4+ and CD8+ T-cellresponses, suggesting a non-essential role for LAG-3, blockade of thePD-1/PD-L1 pathway combined with LAG-3 blockade improved viral controlas compared with PD-L1 blockade alone (Blackburn et al., Nat. Immunol.,10: 29-37 (2009); and Richter et al., Int. Immunol., 22: 13-2 (2010)).

In a self-tolerance/tumor mouse model where transgenic CD8+ T-cells wererendered unresponsive/anergic, in viva, LAG-3 blockade or deficiency inCD8+ T-cells enhanced T-cell proliferation, T-cell recruitment andeffector functions, at the tumor site (Grosso et at, J. Clin. Invest.,117: 3383-92 (2007)).

Inhibition of LAG-3 activity, such as through use of monoclonalantibodies, is currently under investigation as a therapeutic approachto treat viral infections and melanoma based on preclinical studies. Forexample, addition of soluble huLAG-3 fused to an Fc region enhanced theproliferation of antigen-specific T-cells to viral and tumor antigens,such as influenza matrix protein or melanoma antigen recognized byT-cells (MART-1), in PBMCs of healthy or cancer patients (Casati et al.,J. Immunol, 180: 3782-3788 (2008)).

There is a need for additional antagonists of LAG-3 (e.g., an antibody)that binds LAG-3 with high affinity and effectively neutralizes LAG-3activity. The invention provides such LAG-3-binding agents.

BRIEF SUMMARY OF THE INVENTION

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises the amino acid sequence Glu Val Gln Leu ValGln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys LysAla Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln AlaPro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 AspSer Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp ThrSer Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser (SEQ ID NO: 181), wherein (a) Xaa1 is asparagine(Asn) or serine (Ser), (b) Xaa2 is lysine (Lys), tyrosine (Tyr), orasparagine (Asn), (c) Xaa3 is lysine (Lys) or glutamine (Gln), (d) Xaa4is isoleucine (e) or methionine (Met), (e) Xaa5 is alanine (Ala) orproline (Pro), (f) Xaa6 is glutamic acid (Glu) or methionine (Met), (g)Xaa6 is glycine (Gly), asparagine (Asn), or aspartic acid (Asp), (h)Xaa8 is glutamic acid (Glu) or glutamine (Q), (i) Xaa9 is alanine (Ala)or serine (Ser), (j) Xaa10 is glutamine (Gln) or arginine (Arg), (k)Xaa11 is aspartic acid (Asp) or asparagine (Asn), and (1) Xaa12 isglutamine (Gln) or lysine (Lys).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises the amino acid sequence Gln Val Gln Leu GlnGln Trp Gly Ala Xaa1 Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Xaa2 CysXaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly Tyr Tyr Trp Xaa6 Trp

e Arg Pro Xaa7 Lys Gly Leu Glu Trp e Gly Glu e Asn His Ser Gly Xaa8 ThrAsn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr He Ser Val Asp Thr Ser LysAsn Gln Xaa9 Ser Leu Lys Leu Xaa10 Xaa11 Val Thr Ala Ala Asp Thr Ala ValTyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr Gly Asp Tyr Asp Tyr Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser (SEQ ID NO: 35), wherein (a) Xaa1 isarginine (Arg) or glycine (Gly), (b) Xaa2 is threonine (Thr) orisoleucine (Ile), (c) Xaa3 is threonine (Thr) or alanine (Ala), (d) Xaa4is serine (Ser) or phenylalanine (Phe), (e) Xaa5 is serine (Ser) orphenylalanine (Phe), (f) Xaa6 is serine (Ser) or isoleucine (Ile), (g)Xaa7 is glycine (Gly) or arginine (Arg), (h) Xaa8 is serine (Ser) orasparagine (Asn), (i) Xaa9 is phenylalanine (Phe) or leucine (Leu), (j)Xaa10 is asparagine (Asn) or serine (Ser), (k) Xaa11 is serine (Ser) orphenylalanine (Phe), (1) Xaa12 is alanine (Ala) or valine (Val), and (m)Xaa13 is aspartic acid (Asp) or asparagine (Asn).

The invention further provides an isolated immunoglobulin heavy chainpolypeptide comprising SEQ ID NO: 190 or 191.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence Asp Xaa1 Val Met ThrGln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala Ser Ile Ser CysArg Xaa2 Ser Gln Ser Leu Val His Ser Asp Xaa3 Xaa4 Thr Tyr Leu His TipTyr Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Xaa Xaa Ser AsnArg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Lys

e Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gln Ser ThrXaa Val Pro Tyr Ala Phe Gly Gly Gly Thr Lys Val Glu He Lys Arg Thr (SEQID NO: 57), wherein (a) Xaa1 is valine (Val) or isoleucine (Ile), (b)Xaa2 is cysteine (Cys) or serine (Ser), (c) Xaa3 is glycine (Gly) orserine (Ser), (d) Xaa4 is asparagine (Asn) or aspartic acid (Asp),(e)Xaa5 is lysine (Lys), glycine (Gly), asparagine (Asn), serine (Ser),or leucine (Leu), (f) Xaa6 is valine (Val) or isoleucine (le), (g) Xaa7is serine (Ser), alanine (Ala), or glycine (Gly), and (h) Xaa8 ishistidine (His) or tyrosine (Tyr).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises the amino acid sequence Asp Ile Gin Met ThrGln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr CysGln Ala Ser Gln Asp

e Ser Asn Tyr Leu Asn Trp Tyr Gin Gln Lys Pro Gly Lys Ala Pro Lys LeuLeu Ile Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro GluAsp Ile Ala Val Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe GlyGln Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val (SEQ IDNO: 89), wherein (a) the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 is deletedor is Tyr-Asp-Ala-Ser-Asn, and (b) Xaa6 is threonine (Thr) or isoleucine(Ile).

The invention also provides isolated immunoglobulin light chainpolypeptide comprising SEQ ID NO: 196 or 197.

In addition, the invention provides isolated or purified nucleic acidsequences encoding the foregoing immunoglobulin polypeptides, vectorscomprising such nucleic acid sequences, LAG-3-binding agents comprisingthe foregoing immunoglobulin polypeptides, nucleic acid sequencesencoding such LAG-3-binding agents, vectors comprising such nucleic acidsequences, isolated cells comprising such vectors, compositionscomprising such LAG-3-binding agents or such vectors with apharmaceutically acceptable carrier, and methods of treating cancer orinfectious diseases in mammals by administering effective amounts ofsuch compositions to mammals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph of mean tumor volume over time in mice implanted withColon26 colon adenocarcinoma cells and injected with the indicatedantibodies. Each data plot in the figure refers to the indicatedtreatment group.

FIG. 1B is a graph of tumor volume over time of individual animals inthree treatment groups of mice implanted with Colon26 colonadenocarcinoma cells and injected with the indicated antibodies. Eachdata plot in the graphs refers to an individual animal in the treatmentgroup.

FIG. 2A depicts IL-2 secretion by CD4+ T-cells in a mixed lymphocytereaction (MLR) assay at varying concentrations of Anti PD-1 orAnti-LAG-3 antibodies.

FIG. 2B depicts LAG-3 and PD-1 expression on CD4+ T-cells prior to(naïve) or subsequent to (24, 48, and 72 hour) exposure to dendriticcells.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides an isolated immunoglobulin heavy chainpolypeptide and/or an isolated immunoglobulin light chain polypeptide,or a fragment (e.g., antigen-binding fragment) thereof. The term“immunoglobulin” or “antibody,” as used herein, refers to a protein thatis found in blood or other bodily fluids of vertebrates, which is usedby the immune system to identify and neutralize foreign objects, such asbacteria and viruses. The polypeptide is “isolated” in that it isremoved from its natural environment. In a preferred embodiment, animmunoglobulin or antibody is a protein that comprises at least onecomplementarity determining region (CDR). The CDRs form the“hypervariable region” of an antibody, which is responsible for antigenbinding (discussed further below). A whole immunoglobulin typicallyconsists of four polypeptides: two identical copies of a heavy (H) chainpolypeptide and two identical copies of a light (L) chain polypeptide.Each of the heavy chains contains one N-terminal variable (V_(H)) regionand three C-terminal constant (C_(H)1, C_(H)2, and C_(H)3) regions, andeach light chain contains one N-terminal variable (V_(L)) region and oneC-terminal constant (C_(L)) region. The light chains of antibodies canbe assigned to one of two distinct types, either kappa (κ) or lambda(λ), based upon the amino acid sequences of their constant domains. In atypical immunoglobulin, each light chain is linked to a heavy chain bydisulphide bonds, and the two heavy chains are linked to each other bydisulphide bonds. The light chain variable region is aligned with thevariable region of the heavy chain, and the light chain constant regionis aligned with the first constant region of the heavy chain. Theremaining constant regions of the heavy chains are aligned with eachother.

The variable regions of each pair of light and heavy chains form theantigen binding site of an antibody. The V_(H) and V_(L) regions havethe same general structure, with each region comprising four framework(FW or FR) regions. The term “framework region,” as used herein, refersto the relatively conserved amino acid sequences within the variableregion which are located between the hypervariable or complementarydetermining regions (CDRs). There are four framework regions in eachvariable domain, which are designated FR1, FR2, FR3, and FR4. Theframework regions form the β sheets that provide the structuralframework of the variable region (see, e.g., C. A. Janeway et al.(eds.), Immunobiology, 5th Ed., Garland Publishing, New York, N.Y.(2001)).

The framework regions are connected by three complementarity determiningregions (CDRs). As discussed above, the three CDRs, known as CDR1, CDR2,and CDR3, form the “hypervariable region” of an antibody, which isresponsible for antigen binding. The CDRs form loops connecting, and insome cases comprising part of, the beta-sheet structure formed by theframework regions. While the constant regions of the light and heavychains are not directly involved in binding of the antibody to anantigen, the constant regions can influence the orientation of thevariable regions. The constant regions also exhibit various effectorfunctions, such as participation in antibody-dependentcomplement-mediated lysis or antibody-dependent cellular toxicity viainteractions with effector molecules and cells.

The isolated immunoglobulin heavy chain polypeptide and the isolatedimmunoglobulin light chain polypeptide of the invention desirably bindto the protein encoded by the Lymphocyte Activation Gene-3 (LAG-3) (alsoreferred to herein as “LAG-3 protein”). As discussed above, LAG-3 is a498 amino acid protein that negatively regulates T-cell function andhomeostasis (Triebel et al., J. Exp. Med., 171(5): 1393-1405 (1990); andTriebel F., Trends Immunol., 24(12): 619-22 (2003)). LAG-3 is a memberof the immunoglobulin supergene family and is structurally andgenetically related to CD4. The intra-cytoplasmic region of LAG-3 hasbeen shown to interact with a protein denoted LAP, which is thought tobe a signal transduction molecule involved in the downregulation of theCD3/TCR activation pathway (Iouzalen et al., Eur. J. Immunol., 31:2885-2891 (2001)). Furthermore, CD4+CD25+ regulatory T-cells (Treg) havebeen shown to express LAG-3 upon activation and antibodies to LAG-3inhibit suppression by induced Treg cells, both in vitro and in vivo,suggesting that LAG-3 contributes to the suppressor activity of Tregcells (Huang et al., Immunity, 21: 503-513 (2004)). However, a recentstudy suggests that LAG-3 expression on CD4+ T-cells renders them moresusceptible to suppression by Tregs, rather than making Tregs moresuppressive (see Durham et al., PLoS ONE, 9(11): e109080 (2014)). Incertain circumstances, LAG-3 also has been shown to haveimmunostimulatory effects (see, e.g., Prigent et al., Eur. J. Immunol.,29: 3867-3876 (1999)); El Mir and Triebel, J. Immunol., 164: 5583-5589(2000)); and Casati et al., Cancer Res., 66: 4450-4460 (2006)). Theinventive isolated immunoglobulin heavy chain polypeptide and theinventive isolated immunoglobulin light chain polypeptide can form anagent that binds to LAG-3 and another antigen, resulting in a “dualreactive” binding agent (e.g., a dual reactive antibody). For example,the agent can bind to LAG-3 and to another negative regulator of theimmune system such as, for example, programmed death 1 (PD-1) and/orT-cell immunoglobulin domain and mucin domain 3 protein (TIM-3).

Antibodies which bind to LAG-3, and components thereof, are known in theart (see, e.g., U.S. Patent Application Publication Nos. 2010/0233183,2011/0150892, and 2014/0093511). Anti-LAG-3 antibodies also arecommercially available from sources such as, for example, Abcam(Cambridge, Mass.), and R&D Systems, Inc. (Minneapolis, Minn.).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises the amino acid sequence Glu Val Gln Leu ValGln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys LysAla Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr Ile His Trp Val Xaa3 Gln AlaPro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp Ile Asp Xaa5 Xaa6 Asn Xaa7 AspSer Xaa8 Tyr Xaa9 Ser Lys Phe Xaa10 Gly Arg Val Thr Ile Thr Val Asp ThrSer Thr Xaa11 Thr Ala Tyr Met Xaa12 Leu Ser Ser Leu Arg Ser Glu Asp ThrAla Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly Tyr Trp Gly Gln Gly Thr ThrVal Thr Val Ser Ser (SEQ ID NO: 181), wherein (a) Xaa1 is asparagine(Asn) or serine (Ser), (b) Xaa2 is lysine (Lys), tyrosine (Tyr), orasparagine (Asn), (c) Xaa3 is lysine (Lys) or glutamine (Gln), (d) Xaa4is isoleucine (Ile) or methionine (Met), (e) Xaa5 is alanine (Ala) orproline (Pro), (f) Xaa6 is glutamic acid (Glu) or methionine (Met), (g)Xaa6 is glycine (Gly), asparagine (Asn), or aspartic acid (Asp), (h)Xaa8 is glutamic acid (Glu) or glutamine (Q), (i) Xaa9 is alanine (Ala)or serine (Ser), (j) Xaa10 is glutamine (Gln) or arginine (Arg), (k)Xaa11 is aspartic acid (Asp) or asparagine (Asn), and (1) Xaa12 isglutamine (Gln) or lysine (Lys).

In another aspect, the immunoglobulin heavy chain polypeptide comprises,consists of, or consists essentially of the amino acid sequence Glu ValGin Leu Val Gin Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Thr Val Lys IleSer Cys Lys Ala Ser Gly Phe Xaa1 Ile Xaa2 Asp Asp Tyr

e His Trp Val Xaa3 Gin Ala Pro Gly Lys Gly Leu Glu Trp Xaa4 Gly Trp

e Asp Xaa5 Glu Asn Xaa6 Asp Ser Glu Tyr Xaa7 Ser Lys Phe Xaa8 Gly ArgVal Thr Ile Thr Val Asp Thr Ser Thr Xaa9 Thr Ala Tyr Met Glu Leu Ser SerLeu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys Thr Tyr Ala Phe Gly Gly TyrTrp Gly Gin Gly Thr Thr Val Thr Val Ser Ser (SEQ ID NO: 1), wherein (a)Xaa1 is asparagine (Asn) or serine (Ser), (b) Xaa2 is lysine (Lys),tyrosine (Tyr), or asparagine (Asn), (c) Xaa3 is lysine (Lys) orglutamine (Gin), (d) Xaa4 is isoleucine (Ile) or methionine (Met), (e)Xaa5 is alanine (Ala) or proline (Pro), (f) Xaa6 is glycine (Gly),asparagine (Asn), or aspartic acid (Asp), (g) Xaa7 is alanine (Ala) orserine (Ser), (h) Xaa8 is glutamine (Gin) or arginine (Arg), and (i)Xaa9 is aspartic acid (Asp) or asparagine (Asn).

In one embodiment, the isolated immunoglobulin heavy chain polypeptidecomprises, consists of, or consists essentially of an amino acidsequence of any one of SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO: 9, SEQ ID NO:10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24,SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO:29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ IDNO: 34, SEQ ID NO: 182, SEQ ID NO: 183, SEQ ID NO: 184, SEQ ID NO: 185,or SEQ ID NO: 186.

The invention also provides an immunoglobulin heavy chain polypeptidethat comprises, consists of, or consists essentially of the amino acidsequence Gin Val Gin Leu Gin Gin Trp Gly Ala Xaa1 Leu Leu Lys Pro SerGlu Thr Lu Ser Leu Xaa2 Cys Xaa3 Val Tyr Gly Gly Xaa4 Phe Xaa5 Gly TyrTyr Trp Xaa6 Trp Ile Arg Gin Pro Pro Xaa7 Lys Gly Leu Glu Trp Ile GlyGlu Ile Asn His Ser Gly Xaa8 Thr Asn Tyr Asn Pro Ser Leu Lys Ser Arg ValThr

e Ser Val Asp Thr Ser Lys Asn Gin Xaa9 Ser Leu Lys Leu Xaa10 Xaa11 ValThr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Xaa12 Arg Glu Gly Xaa13 Tyr GlyAsp Tyr Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser (SEQ ID NO:35), wherein (a) Xaa1 is arginine (Arg) or glycine (Gly), (b) Xaa2 isthreonine (Thr) or isoleucine (Ile), (c) Xaa3 is threonine (Thr) oralanine (Ala), (d) Xaa4 is serine (Ser) or phenylalanine (Phe), (e) Xaa5is serine (Ser) or phenylalanine (Phe), (f) Xaa6 is serine (Ser) orisoleucine (Ile), (g) Xaa7 is glycine (Gly) or arginine (Arg), (h) Xaa8is serine (Ser) or asparagine (Asn), (i) Xaa9 is phenylalanine (Phe) orleucine (Leu), (j) Xaa10 is asparagine (Asn) or serine (Ser), (k) Xaa11is serine (Ser) or phenylalanine (Phe), (1) Xaa12 is alanine (Ala) orvaline (Val), and (m) Xaa13 is aspartic acid (Asp) or asparagine (Asn).

In one embodiment, the isolated immunoglobulin heavy chain polypeptidecomprises, consists of, or consists essentially of an amino acidsequence of any one of SEQ ID NO: 36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ IDNO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.

In another embodiment, there is provided an isolated immunoglobulinheavy chain polypeptide which comprises SEQ ID NO: 190 or 191. Examplesof such a polypeptide include those comprising any one of SEQ ID NOs:192-195.

When the inventive immunoglobulin heavy chain polypeptide consistsessentially of an amino acid sequence of any one of SEQ ID NO: 1-SEQ IDNO: 56, SEQ ID NOS: 182-186, or SEQ ID NOS: 190-195, additionalcomponents can be included in the polypeptide that do not materiallyaffect the polypeptide (e.g., protein moieties such as biotin thatfacilitate purification or isolation). When the inventive immunoglobulinheavy chain polypeptide consists of an amino acid sequence of any one ofSEQ ID NO: 1-SEQ ID NO: 56, the polypeptide does not comprise anyadditional components (i.e., components that are not endogenous to theinventive immunoglobulin heavy chain polypeptide).

The invention provides an isolated immunoglobulin heavy chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 1-56. Nucleic acid or aminoacid sequence “identity,” as described herein, can be determined bycomparing a nucleic acid or amino acid sequence of interest to areference nucleic acid or amino acid sequence. The percent identity isthe number of nucleotides or amino acid residues that are the same(i.e., that are identical) as between the sequence of interest and thereference sequence divided by the length of the longest sequence (i.e.,the length of either the sequence of interest or the reference sequence,whichever is longer). A number of mathematical algorithms for obtainingthe optimal alignment and calculating identity between two or moresequences are known and incorporated into a number of available softwareprograms. Examples of such programs include CLUSTAL-W, T-Coffee, andALIGN (for alignment of nucleic acid and amino acid sequences). BLASTprograms (e.g., BLAST 2.1, BL2SEQ, and later versions thereof) and FASTAprograms (e.g., FASTA3x, FASTM, and SSEARCH) (for sequence alignment andsequence similarity searches). Sequence alignment algorithms also aredisclosed in, for example, Altschul et al., J. Molecular Biol., 215(3):403-410 (1990), Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis:Probalistic Models of Proteins and Nucleic Acids, Cambridge UniversityPress, Cambridge, UK (2009), Soding, Bioinformatics, 21(7): 951-960(2005), Altschul et al., Nucleic Acids Res., 25(17): 3389-3402 (1997),and Gusfield, Algorithms on Strings, Trees and Sequences, CambridgeUniversity Press, Cambridge UK (1997)).

In another embodiment, the invention provides an immunoglobulin lightchain polypeptide that comprises, consists of, or consists essentiallyof, an isolated immunoglobulin light chain polypeptide which comprisesthe amino acid sequence Asp Xaa1 Val Met Thr Gln Thr Pro Leu Ser Leu SerVal Thr Pro Gly Gin Pro Ala Ser Ile Ser Cys Arg Xaa2 Ser Gln Ser Leu ValHis Ser Asp Xaa3 Xaa4 Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly GinSer Pro Gin Leu Leu Ile Tyr Xaa Xaa Ser Asn Arg Phe Ser Gly Val Pro AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg ValGlu Ala Glu Asp Val Gly Val Tyr Phe Cys Xaa Gin Ser Thr Xaa Val Pro TyrAla Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr (SEQ ID NO: 57),wherein (a) Xaa1 is valine (Val) or isoleucine (

e), (b) Xaa2 is cysteine (Cys) or serine (Ser), (c) Xaa3 is glycine(Gly) or serine (Ser), (d) Xaa4 is asparagine (Asn) or aspartic acid(Asp), (e)Xaa5 is lysine (Lys), glycine (Gly), asparagine (Asn), serine(Ser), or leucine (Leu), (f) Xaa6 is valine (Val) or isoleucine (Ile),(g) Xaa7 is serine (Ser), alanine (Ala), or glycine (Gly), and (h) Xaa8is histidine (His) or tyrosine (Tyr).

In one embodiment, the isolated immunoglobulin light chain polypeptidecomprises, consists of, or consists essentially of an amino acidsequence of any one of SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQID NO: 61. SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64. SEQ ID NO: 65,SEQ ID NO: 66, SEQ ID NO: 67. SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO:70. SEQ ID NO: 71. SEQ ID NO: 72, SEQ ID NO: 73. SEQ ID NO: 74. SEQ IDNO: 75, SEQ ID NO: 76. SEQ ID NO: 77. SEQ ID NO: 78. SEQ ID NO: 79. SEQID NO: 80. SEQ ID NO: 81. SEQ ID NO: 82. SEQ ID NO: 83. SEQ ID NO: 84,SEQ ID NO: 85. SEQ ID NO: 86. SEQ ID NO: 87. SEQ ID NO: 88. SEQ ID NO:187. SEQ ID NO: 188, or SEQ ID NO: 189.

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises, consists essentially of, or consists of theamino acid sequence Asp Ile Gin Met Thr Gln Ser Pro Ser Ser Leu Ser AlaSer Val Gly Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser AsnTyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Xaa1Xaa2 Xaa3 Xaa4 Xaa5 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile AlaVal Tyr Tyr Cys Gln Gln Ser Tyr Ser Xaa6 Leu Ile Thr Phe Gly Gln Gly ThrArg Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val (SEQ ID NO: 89),wherein (a) the subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 is deleted or isTyr-Asp-Ala-Ser-Asn, and (b) Xaa6 is threonine (Thr) or isoleucine(Ile).

The inventive immunoglobulin light chain polypeptide can include or lackthe subsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 at positions 49-53 of SEQ IDNO: 89 when Xaa6 is threonine (Thr) or isoleucine (Ile). When theinventive immunoglobulin light chain polypeptide comprises thesubsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5, each of Xaa1, Xaa2, Xaa3, Xaa4,and Xaa5 can be any suitable amino acid residue. Preferably. Xaa1 istyrosine (Tyr), Xaa2 is aspartic acid (Asp). Xaa3 is alanine (Ala), Xaa4is serine (Ser), and Xaa5 is asparagine (Asn). A preferred amino acidsequence of an immunoglobulin light chain polypeptide which includes thesubsequence Xaa1 Xaa2 Xaa3 Xaa4 Xaa5 comprises SEQ ID NO: 90. When theimmunoglobulin light chain polypeptide lacks the subsequence Xaa1 Xaa2Xaa3 Xaa4 Xaa5, the immunoglobulin light chain polypeptide preferablycomprises the amino acid sequence SEQ ID NO: 91 or SEQ ID NO: 92.

In another embodiment, provided is an isolated immunoglobulin lightchain polypeptide which comprises SEQ ID NO: 196 or 197. Examples ofsuch a polypeptide include those comprising any one of SEQ ID NOs:198-200.

When the inventive immunoglobulin light chain polypeptide consistsessentially of an amino acid sequence of any one of SEQ ID NO: 57-SEQ IDNO: 92, SEQ ID NOs: 187-189, or SEQ ID NOs: 196-200, additionalcomponents can be included in the polypeptide that do not materiallyaffect the polypeptide (e.g., protein moieties such as biotin thatfacilitate purification or isolation). When the inventive immunoglobulinlight chain polypeptide consists of an amino acid sequence of any one ofSEQ ID NO: 57-SEQ ID NO: 92, the polypeptide does not comprise anyadditional components (i.e., components that are not endogenous to theinventive immunoglobulin light chain polypeptide).

The invention provides an isolated immunoglobulin light chainpolypeptide which comprises an amino acid sequence that is at least 90%identical (e.g., at least 91%, at least 92%, at least 93%, at least 94%,at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100% identical) to any one of SEQ ID NO: 57-SEQ ID NO: 92. Nucleic acidor amino acid sequence “identity” can be determined using the methodsdescribed herein.

One or more amino acids of the aforementioned immunoglobulin heavy chainpolypeptides and/or light chain polypeptides can be replaced orsubstituted with a different amino acid. An amino acid “replacement” or“substitution” refers to the replacement of one amino acid at a givenposition or residue by another amino acid at the same position orresidue within a polypeptide sequence.

Amino acids are broadly grouped as “aromatic” or “aliphatic.” Anaromatic amino acid includes an aromatic ring. Examples of “aromatic”amino acids include histidine (H or His), phenylalanine (F or Phe),tyrosine (Y or Tyr), and tryptophan (W or Trp). Non-aromatic amino acidsare broadly grouped as “aliphatic.” Examples of “aliphatic” amino acidsinclude glycine (G or Gly), alanine (A or Ala), valine (V or Val),leucine (L or Leu), isoleucine (I or Ile), methionine (M or Met), serine(S or Ser), threonine (T or Thr), cysteine (C or Cys), proline (P orPro), glutamic acid (E or Glu), aspartic acid (A or Asp), asparagine (Nor Asn), glutamine (Q or Gln), lysine (K or Lys), and arginine (R orArg).

Aliphatic amino acids may be sub-divided into four sub-groups. The“large aliphatic non-polar sub-group” consists of valine, leucine, andisoleucine. The “aliphatic slightly-polar sub-group” consists ofmethionine, serine, threonine, and cysteine. The “aliphaticpolar/charged sub-group” consists of glutamic acid, aspartic acid,asparagine, glutamine, lysine, and arginine. The “small-residuesub-group” consists of glycine and alanine. The group of charged/polaramino acids may be sub-divided into three sub-groups: the“positively-charged sub-group” consisting of lysine and arginine, the“negatively-charged sub-group” consisting of glutamic acid and asparticacid, and the “polar sub-group” consisting of asparagine and glutamine.

Aromatic amino acids may be sub-divided into two sub-groups: the“nitrogen ring sub-group” consisting of histidine and tryptophan and the“phenyl sub-group” consisting of phenylalanine and tyrosine.

The amino acid replacement or substitution can be conservative,semi-conservative, or non-conservative. The phrase “conservative aminoacid substitution” or “conservative mutation” refers to the replacementof one amino acid by another amino acid with a common property. Afunctional way to define common properties between individual aminoacids is to analyze the normalized frequencies of amino acid changesbetween corresponding proteins of homologous organisms (Schulz andSchirmer, Principles of Protein Structure, Springer-Verlag, New York(1979)). According to such analyses, groups of amino acids may bedefined where amino acids within a group exchange preferentially witheach other, and therefore resemble each other most in their impact onthe overall protein structure (Schulz and Schirmer, supra).

Examples of conservative amino acid substitutions include substitutionsof amino acids within the sub-groups described above, for example,lysine for arginine and vice versa such that a positive charge may bemaintained, glutamic acid for aspartic acid and vice versa such that anegative charge may be maintained, serine for threonine such that a free—OH can be maintained, and glutamine for asparagine such that a free—NH₂ can be maintained.

“Semi-conservative mutations” include amino acid substitutions of aminoacids within the same groups listed above, but not within the samesub-group. For example, the substitution of aspartic acid forasparagine, or asparagine for lysine, involves amino acids within thesame group, but different sub-groups. “Non-conservative mutations”involve amino acid substitutions between different groups, for example,lysine for tryptophan, or phenylalanine for serine, etc.

In addition, one or more amino acids can be inserted into theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides. Any number of any suitable amino acids can beinserted into the amino acid sequence of the immunoglobulin heavy chainpolypeptide and/or light chain polypeptide. In this respect, at leastone amino acid (e.g., 2 or more, 5 or more, or 10 or more amino acids),but not more than 20 amino acids (e.g., 18 or less, 15 or less, or 12 orless amino acids), can be inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.Preferably, 1-10 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10amino acids) are inserted into the amino acid sequence of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.In this respect, the amino acid(s) can be inserted into any one of theaforementioned immunoglobulin heavy chain polypeptides and/or lightchain polypeptides in any suitable location. Preferably, the aminoacid(s) are inserted into a CDR (e.g., CDR1, CDR2, or CDR3) of theimmunoglobulin heavy chain polypeptide and/or light chain polypeptide.

The inventive isolated immunoglobulin heavy chain polypeptide and lightchain polypeptides are not limited to polypeptides comprising thespecific amino acid sequences described herein. Indeed, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that competeswith the inventive immunoglobulin heavy chain polypeptide or light chainpolypeptide for binding to LAG-3. In this respect, for example, theimmunoglobulin heavy chain polypeptide or light chain polypeptide can beany heavy chain polypeptide or light chain polypeptide that binds to thesame epitope of LAG-3 recognized by the heavy and light chainpolypeptides described herein. Antibody competition can be assayed usingroutine peptide competition assays which utilize ELISA, Western blot, orimmunohistochemistry methods (see, e.g., U.S. Pat. Nos. 4,828,981 and8,568,992; and Braitbard et al., Proteome Sci., 4: 12 (2006)).

The invention provides an isolated LAG-3-binding agent comprising,consisting essentially of, or consisting of one or more of the inventiveisolated amino acid sequences described herein. By “LAG-3-binding agent”is meant a molecule, preferably a proteinaceous molecule, which bindsspecifically to the LAG-3 protein. Preferably, the LAG-3-binding agentis an antibody or a fragment (e.g., immunogenic fragment) thereof. TheLAG-3-binding agent of the invention comprises, consists essentially of,or consists of the inventive isolated immunoglobulin heavy chainpolypeptide and/or the inventive isolated immunoglobulin light chainpolypeptide. In one embodiment, the LAG-3-binding agent comprises,consists essentially of, or consists of the inventive immunoglobulinheavy chain polypeptide or the inventive immunoglobulin light chainpolypeptide. In another embodiment, the LAG-3-binding agent comprises,consists essentially of, or consists of the inventive immunoglobulinheavy chain polypeptide and the inventive immunoglobulin light chainpolypeptide.

Any amino acid residue of the inventive immunoglobulin heavy chainpolypeptide and/or the inventive immunoglobulin light chain polypeptidecan be replaced, in any combination, with a different amino acidresidue, or can be deleted or inserted, so long as the biologicalactivity of the LAG-3-binding agent is enhanced or improved as a resultof the amino acid replacements, insertions, and/or deletions. The“biological activity” of an LAG-3-binding agent refers to, for example,binding affinity for a particular LAG-3 epitope, neutralization orinhibition of LAG-3 binding to its receptor(s), neutralization orinhibition of LAG-3 activity in vivo (e.g., IC₅₀), pharmacokinetics, andcross-reactivity (e.g., with non-human homologs or orthologs of theLAG-3 protein, or with other proteins or tissues). Other biologicalproperties or characteristics of an antigen-binding agent recognized inthe art include, for example, avidity, selectivity, solubility, folding,immunotoxicity, expression, and formulation. The aforementionedproperties or characteristics can be observed, measured, and/or assessedusing standard techniques including, but not limited to, ELISA,competitive ELISA, surface plasmon resonance analysis (BIACORE™), orKINEXA™, in vitro or in vivo neutralization assays, receptor-ligandbinding assays, cytokine or growth factor production and/or secretionassays, and signal transduction and immunohistochemistry assays.

The terms “inhibit” or “neutralize,” as used herein with respect to theactivity of a LAG-3-binding agent, refer to the ability to substantiallyantagonize, prohibit, prevent, restrain, slow, disrupt, alter,eliminate, stop, or reverse the progression or severity of, for example,the biological activity of LAG-3, or a disease or condition associatedwith LAG-3. The isolated LAG-3-binding agent of the invention preferablyinhibits or neutralizes the activity of LAG-3 by at least about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 100%, or a range defined by any two of theforegoing values.

The isolated LAG-3-binding agent of the invention can be a wholeantibody, as described herein, or an antibody fragment. The terms“fragment of an antibody,” “antibody fragment,” and “functional fragmentof an antibody” are used interchangeably herein to mean one or morefragments of an antibody that retain the ability to specifically bind toan antigen (see, generally, Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). The isolated LAG-3-binding agent can contain anyLAG-3-binding antibody fragment. The antibody fragment desirablycomprises, for example, one or more CDRs, the variable region (orportions thereof), the constant region (or portions thereof), orcombinations thereof. Examples of antibody fragments include, but arenot limited to, (i) a Fab fragment, which is a monovalent fragmentconsisting of the V_(L), V_(H), C_(L), and CH₁ domains, (ii) a F(ab′)₂fragment, which is a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region, (iii) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (iv) a Fab′ fragment, which results from breaking thedisulfide bridge of an F(ab′)₂ fragment using mild reducing conditions,(v) a disulfide-stabilized Fv fragment (dsFv), and (vi) a domainantibody (dAb), which is an antibody single variable region domain (VHor VL) polypeptide that specifically binds antigen.

In embodiments where the isolated LAG-3-binding agent comprises afragment of the immunoglobulin heavy chain or light chain polypeptide,the fragment can be of any size so long as the fragment binds to, andpreferably inhibits the activity of, LAG-3. In this respect, a fragmentof the immunoglobulin heavy chain polypeptide desirably comprisesbetween about 5 and 18 (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, or a range defined by any two of the foregoing values)amino acids. Similarly, a fragment of the immunoglobulin light chainpolypeptide desirably comprises between about 5 and 18 (e.g., about 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined byany two of the foregoing values) amino acids.

When the LAG-3-binding agent is an antibody or antibody fragment, theantibody or antibody fragment desirably comprises a heavy chain constantregion (Fe) of any suitable class. Preferably, the antibody or antibodyfragment comprises a heavy chain constant region that is based uponwild-type IgG1, IgG2, or IgG4 antibodies, or variants thereof. In someembodiments, the LAG-3 binding agent comprises an Fc region engineeredto reduce or eliminate effector functions of the antibody. Engineered Fcregions with reduced or abrogated effector function are known in the artand commercially available, as are techniques for engineering Fc regionsto reduce or eliminate effector function, any of which can be used inconjunction with the invention.

The LAG-3-binding agent also can be a single chain antibody fragment.Examples of single chain antibody fragments include, but are not limitedto, (i) a single chain Fv (scFv), which is a monovalent moleculeconsisting of the two domains of the Fv fragment (i.e., V_(L) and VH)joined by a synthetic linker which enables the two domains to besynthesized as a single polypeptide chain (see, e.g., Bird et al.,Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA,85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778(1998)) and (ii) a diabody, which is a dimer of polypeptide chains,wherein each polypeptide chain comprises a V_(H) connected to a V_(L) bya peptide linker that is too short to allow pairing between the V_(H)and V_(L) on the same polypeptide chain, thereby driving the pairingbetween the complementary domains on different V_(H)-V_(L) polypeptidechains to generate a dimeric molecule having two functional antigenbinding sites. Antibody fragments are known in the art and are describedin more detail in, e.g., U.S. Patent Application Publication2009/0093024 A1.

The isolated LAG-3-binding agent also can be an intrabody or fragmentthereof. An intrabody is an antibody which is expressed and whichfunctions intracellularly. Intrabodies typically lack disulfide bondsand are capable of modulating the expression or activity of target genesthrough their specific binding activity. Intrabodies include singledomain fragments such as isolated V_(H) and V_(L) domains and scFvs. Anintrabody can include sub-cellular trafficking signals attached to the Nor C terminus of the intrabody to allow expression at highconcentrations in the sub-cellular compartments where a target proteinis located. Upon interaction with a target gene, an intrabody modulatestarget protein function and/or achieves phenotypic/functional knockoutby mechanisms such as accelerating target protein degradation andsequestering the target protein in a non-physiological sub-cellularcompartment. Other mechanisms of intrabody-mediated gene inactivationcan depend on the epitope to which the intrabody is directed, such asbinding to the catalytic site on a target protein or to epitopes thatare involved in protein-protein, protein-DNA, or protein-RNAinteractions.

The isolated LAG-3-binding agent also can be an antibody conjugate. Inthis respect, the isolated LAG-3-binding agent can be a conjugate of (1)an antibody, an alternative scaffold, or fragments thereof, and (2) aprotein or non-protein moiety comprising the LAG-3-binding agent. Forexample, the LAG-3-binding agent can be all or part of an antibodyconjugated to a peptide, a fluorescent molecule, or a chemotherapeuticagent.

The isolated LAG-3-binding agent can be, or can be obtained from, ahuman antibody, a non-human antibody, or a chimeric antibody. By“chimeric” is meant an antibody or fragment thereof comprising bothhuman and non-human regions. Preferably, the isolated LAG-3-bindingagent is a humanized antibody. A “humanized” antibody is a monoclonalantibody comprising a human antibody scaffold and at least one CDRobtained or derived from a non-human antibody. Non-human antibodiesinclude antibodies isolated from any non-human animal, such as, forexample, a rodent (e.g., a mouse or rat). A humanized antibody cancomprise, one, two, or three CDRs obtained or derived from a non-humanantibody. In one embodiment of the invention, CDRH3 of the inventiveLAG-3-binding agent is obtained or derived from a mouse monoclonalantibody, while the remaining variable regions and constant region ofthe inventive LAG-3-binding agent are obtained or derived from a humanmonoclonal antibody.

A human antibody, a non-human antibody, a chimeric antibody, or ahumanized antibody can be obtained by any means, including via in vitrosources (e.g., a hybridoma or a cell line producing an antibodyrecombinantly) and in vivo sources (e.g., rodents). Methods forgenerating antibodies are known in the art and are described in, forexample, Köhler and Milstein, Eur. J. Immunol., 5: 511-519 (1976);Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press(1988); and Janeway et al. (eds.), Immunobiology, 5th Ed., GarlandPublishing, New York, N.Y. (2001)). In certain embodiments, a humanantibody or a chimeric antibody can be generated using a transgenicanimal (e.g., a mouse) wherein one or more endogenous immunoglobulingenes are replaced with one or more human immunoglobulin genes. Examplesof transgenic mice wherein endogenous antibody genes are effectivelyreplaced with human antibody genes include, but are not limited to, theMedarex HUMAB-MOUSE™, the Kirin TC MOUSE™, and the Kyowa Kirin KM-MOUSE™(see, e.g., Lonberg, Nat. Biotechnol., 23(9): 1117-25 (2005), andLonberg, Handb. Exp. Pharmacol., 181: 69-97 (2008)). A humanizedantibody can be generated using any suitable method known in the art(see, e.g., An, Z. (ed.), Therapeutic Monoclonal Antibodies: From Benchto Clinic, John Wiley & Sons. Inc., Hoboken, N.J. (2009)), including,e.g., grafting of non-human CDRs onto a human antibody scaffold (see,e.g., Kashmiri et al., Methods, 36(1): 25-34 (2005); and Hou et al., J.Biochem., 144(1): 115-120 (2008)). In one embodiment, a humanizedantibody can be produced using the methods described in, e.g., U.S.Patent Application Publication 2011/0287485 A1.

In one embodiment, a CDR (e.g., CDR1, CDR2, or CDR3) or a variableregion of the immunoglobulin heavy chain polypeptide and/or theimmunoglobulin light chain polypeptide described herein can betransplanted (i.e., grafted) into another molecule, such as an antibodyor non-antibody polypeptide, using either protein chemistry orrecombinant DNA technology. In this regard, the invention provides anisolated LAG-3-binding agent comprising at least one CDR of animmunoglobulin heavy chain and/or light chain polypeptide as describedherein. The isolated LAG-3-binding agent can comprise one, two, or threeCDRs of an immunoglobulin heavy chain and/or light chain variable regionas described herein.

In a preferred embodiment, the LAG-3-binding agent binds an epitope ofLAG-3 which blocks the binding of LAG-3 to MHC Class II molecules andinhibits LAG-3-mediated signaling. For example, the LAG-3 binding agentcan bind to one or more of the four Ig-like extracellular domains(D1-D4) of the LAG-3 protein (see, e.g, Triebel et al., J. Exp. Med.,171(5): 1393-1405 (1990); and Bruniquel et al., Immunogenetics, 47:96-98 (1997)). Preferably, the LAG-3 binding agent binds to domain 1(D1) and/or domain (D2) of the LAG-3 protein. The invention alsoprovides an isolated or purified epitope of LAG-3 which blocks thebinding of LAG-3 to MHC Class II molecules in an indirect or allostericmanner.

The invention also provides one or more isolated or purified nucleicacid sequences that encode the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide, andthe inventive LAG-3-binding agent.

The term “nucleic acid sequence” is intended to encompass a polymer ofDNA or RNA, i.e., a polynucleotide, which can be single-stranded ordouble-stranded and which can contain non-natural or alterednucleotides. The terms “nucleic acid” and “polynucleotide” as usedherein refer to a polymeric form of nucleotides of any length, eitherribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms referto the primary structure of the molecule, and thus include double- andsingle-stranded DNA, and double- and single-stranded RNA. The termsinclude, as equivalents, analogs of either RNA or DNA made fromnucleotide analogs and modified polynucleotides such as, though notlimited to, methylated and/or capped polynucleotides. Nucleic acids aretypically linked via phosphate bonds to form nucleic acid sequences orpolynucleotides, though many other linkages are known in the art (e.g.,phosphorothioates, boranophosphates, and the like).

The invention further provides a vector comprising one or more nucleicacid sequences encoding the inventive immunoglobulin heavy chainpolypeptide, the inventive immunoglobulin light chain polypeptide,and/or the inventive LAG-3-binding agent. The vector can be, forexample, a plasmid, episome, cosmid, viral vector (e.g., retroviral oradenoviral), or phage. Suitable vectors and methods of vectorpreparation are well known in the art (see, e.g., Sambrook et al.,Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, New York, N.Y. (1994)).

In addition to the nucleic acid sequence encoding the inventiveimmunoglobulin heavy polypeptide, the inventive immunoglobulin lightchain polypeptide, and/or the inventive LAG-3-binding agent, the vectorpreferably comprises expression control sequences, such as promoters,enhancers, polyadenylation signals, transcription terminators, signalpeptides (e.g., the osteonectin signal peptide), internal ribosome entrysites (IRES), and the like, that provide for the expression of thecoding sequence in a host cell. Exemplary expression control sequencesare known in the art and described in, for example, Goeddel, GeneExpression Technology: Methods in Enzymology, Vol. 185, Academic Press,San Diego, Calif. (1990).

A large number of promoters, including constitutive, inducible, andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources of promoters include forexample, virus, mammal, insect, plant, yeast, and bacteria, and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available, for example, fromdepositories such as the ATCC as well as other commercial or individualsources. Promoters can be unidirectional (i.e., initiate transcriptionin one direction) or bi-directional (i.e., initiate transcription ineither a 3′ or 5′ direction). Non-limiting examples of promotersinclude, for example, the T7 bacterial expression system, pBAD (araA)bacterial expression system, the cytomegalovirus (CMV) promoter, theSV40 promoter, the RSV promoter. Inducible promoters include, forexample, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), theEcdysone inducible system (No et al., Proc. Natl. Acad. Sci., 93:3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.),LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERTtamoxifen inducible recombinase system (Indra et al., Nuc. Acid. Res.,27: 4324-4327 (1999); Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No.7,112,715; and Kramer & Fussenegger, Methods Mol. Biol, 308: 123-144(2005)).

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a nucleic acid sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding region of the nucleic acid sequence and can mediatethe binding of regulatory factors, patterns of DNA methylation, orchanges in DNA structure. A large number of enhancers from a variety ofdifferent sources are well known in the art and are available as orwithin cloned polynucleotides (from, e.g., depositories such as the ATCCas well as other commercial or individual sources). A number ofpolynucleotides comprising promoters (such as the commonly-used CMVpromoter) also comprise enhancer sequences. Enhancers can be locatedupstream, within, or downstream of coding sequences.

The vector also can comprise a “selectable marker gene.” The term“selectable marker gene,” as used herein, refers to a nucleic acidsequence that allow cells expressing the nucleic acid sequence to bespecifically selected for or against, in the presence of a correspondingselective agent. Suitable selectable marker genes are known in the artand described in, e.g., International Patent Application Publications WO1992/008796 and WO 1994/028143; Wigler et al., Proc. Natl. Acad. Sci.USA, 77: 3567-3570 (1980); O'Hare et al., Proc. Natl. Aced. Sci. USA,78: 1527-1531(1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072-2076 (1981); Colberre-Garapin et al., J. Mol. Biol., 150: 1-14(1981); Santerre et al., Gene, 30: 147-156 (1984); Kent et al., Science,237: 901-903 (1987); Wigler et al., Cell, 11: 223-232 (1977); Szybalska& Szybalski, Proc. Natl. Aced. Sci. USA, 48: 2026-2034 (1962); Lowy etal., Cell, 22: 817-823 (1980); and U.S. Pat. Nos. 5,122,464 and5,770,359.

In some embodiments, the vector is an “episomal expression vector” or“episome,” which is able to replicate in a host cell, and persists as anextrachromosomal segment of DNA within the host cell in the presence ofappropriate selective pressure (see, e.g., Conese et al., Gene Therapy,11: 1735-1742 (2004)). Representative commercially available episomalexpression vectors include, but are not limited to, episomal plasmidsthat utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and the Epstein BarrVirus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4,pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.) and pBK-CMV fromStratagene (La Jolla, Calif.) represent non-limiting examples of anepisomal vector that uses T-antigen and the SV40 origin of replicationin lieu of EBNA1 and oriP.

Other suitable vectors include integrating expression vectors, which mayrandomly integrate into the host cell's DNA, or may include arecombination site to enable the specific recombination between theexpression vector and the host cell's chromosome. Such integratingexpression vectors may utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the fip-in system fromInvitrogen (Carlsbad, Calif.) (e.g., pcDNA™5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene (La Jolla, Calif.). Examples of vectors that randomlyintegrate into host cell chromosomes include, for example, pcDNA3.1(when introduced in the absence of T-antigen) from Life Technologies(Carlsbad, Calif.), UCOE from Millipore (Billerica, Mass.), and pCI orpFN10A (ACT) FLEXI™ from Promega (Madison, Wis.).

Viral vectors also can be used. Representative commercially availableviral expression vectors include, but are not limited to, theadenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, TheNetherlands), the lentiviral-based pLP1 from Invitrogen (Carlsbad,Calif.), and the retroviral vectors pFB-ERV plus pCFB-EGSH fromStratagene (La Jolla, Calif.).

Nucleic acid sequences encoding the inventive amino acid sequences canbe provided to a cell on the same vector (i.e., in cis). Aunidirectional promoter can be used to control expression of eachnucleic acid sequence. In another embodiment, a combination ofbidirectional and unidirectional promoters can be used to controlexpression of multiple nucleic acid sequences. Nucleic acid sequencesencoding the inventive amino acid sequences alternatively can beprovided to the population of cells on separate vectors (i.e., intrans). Each of the nucleic acid sequences in each of the separatevectors can comprise the same or different expression control sequences.The separate vectors can be provided to cells simultaneously orsequentially.

The vector(s) comprising the nucleic acid(s) encoding the inventiveamino acid sequences can be introduced into a host cell that is capableof expressing the polypeptides encoded thereby, including any suitableprokaryotic or eukaryotic cell. As such, the invention provides anisolated cell comprising the inventive vector. Preferred host cells arethose that can be easily and reliably grown, have reasonably fast growthrates, have well characterized expression systems, and can betransformed or transfected easily and efficiently.

Examples of suitable prokaryotic cells include, but are not limited to,cells from the genera Bacillus (such as Bacillus subtilis and Bacillusbrevis), Escherichia (such as E. coli), Pseudomonas, Streptomyces,Salmonella, and Erwinia. Particularly useful prokaryotic cells includethe various strains of Escherichia coli (e.g., K12, HB101 (ATCC No.33694), DH5a, DH10, MC1061 (ATCC No. 53338), and CC102).

Preferably, the vector is introduced into a eukaryotic cell. Suitableeukaryotic cells are known in the art and include, for example, yeastcells, insect cells, and mammalian cells. Examples of suitable yeastcells include those from the genera Kluyveromyces, Pichia,Rhino-sporidium, Saccharomyces, and Schizosaccharomyces. Preferred yeastcells include, for example, Saccharomyces cerivisae and Pichia pastoris.

Suitable insect cells are described in, for example, Kitts et al.,Biotechniques, 14: 810-817 (1993); Lucklow, Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al., J. Virol., 67: 4566-4579 (1993).Preferred insect cells include Sf-9 and HI5 (Invitrogen, Carlsbad,Calif.).

Preferably, mammalian cells are utilized in the invention. A number ofsuitable mammalian host cells are known in the art, and many areavailable from the American Type Culture Collection (ATCC, Manassas,Va.). Examples of suitable mammalian cells include, but are not limitedto, Chinese hamster ovary cells (CHO)(ATCC No. CCL61), CHO DHFR-cells(Urlaub et al., Proc. Natl. Acad. Sci. USA, 97: 42164220 (1980)), humanembryonic kidney (HEK) 293 or 293T cells (ATCC No. CRL1573), and 3T3cells (ATCC No. CCL92). Other suitable mammalian cell lines are themonkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCC No. CRL1651),as well as the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Other suitable mammalian cell linesinclude, but are not limited to, mouse neuroblastoma N2A cells, HeLa,mouse L-929 cells, and BHK or HaK hamster cell lines, all of which areavailable from the ATCC. Methods for selecting suitable mammalian hostcells and methods for transformation, culture, amplification, screening,and purification of cells are known in the art.

In one embodiment, the mammalian cell is a human cell. For example, themammalian cell can be a human lymphoid or lymphoid derived cell line,such as a cell line of pre-B lymphocyte origin. Examples of humanlymphoid cells lines include, without limitation, RAMOS (CRL-1596),Daudi (CCL-213), EB-3 (CCL-85), DT40 (CRL-2111), 18-81 (Jack et al.,Proc. Natl. Acad. Sci. USA, 85:1581-1585 (1988)), Raji cells (CCL-86),PER.C6 cells (Crucell Holland B.V., Leiden, The Netherlands), andderivatives thereof.

A nucleic acid sequence encoding the inventive amino acid sequence maybe introduced into a cell by “transfection,” “transformation,” or“transduction.” “Transfection,” “transformation.” or “transduction,” asused herein, refer to the introduction of one or more exogenouspolynucleotides into a host cell by using physical or chemical methods.Many transfection techniques are known in the art and include, forexample, calcium phosphate DNA co-precipitation (see, e.g., Murray E. J.(ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer andExpression Protocols, Humana Press (1991)); DEAE-dextran;electroporation; cationic liposome-mediated transfection; tungstenparticle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash etal., Mol. Cell Biol., 7: 2031-2034 (1987)). Phage or viral vectors canbe introduced into host cells, after growth of infectious particles insuitable packaging cells, many of which are commercially available.

The invention provides a composition comprising an effective amount ofthe inventive immunoglobulin heavy chain polypeptide, the inventiveimmunoglobulin light chain polypeptide, the inventive LAG-3-bindingagent, the inventive nucleic acid sequence encoding any of theforegoing, or the inventive vector comprising the inventive nucleic acidsequence. Preferably, the composition is a pharmaceutically acceptable(e.g., physiologically acceptable) composition, which comprises acarrier, preferably a pharmaceutically acceptable (e.g., physiologicallyacceptable) carrier, and the inventive amino acid sequences,antigen-binding agent, or vector. Any suitable carrier can be usedwithin the context of the invention, and such carriers are well known inthe art. The choice of carrier will be determined, in part, by theparticular site to which the composition may be administered and theparticular method used to administer the composition. The compositionoptionally can be sterile. The composition can be frozen or lyophilizedfor storage and reconstituted in a suitable sterile carrier prior touse. The compositions can be generated in accordance with conventionaltechniques described in, e.g., Remington: The Science and Practice ofPharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, Pa.(2001).

The invention further provides a method of treating a disorder in amammal that is responsive to LAG-3 inhibition or neutralization. Themethod comprises administering the aforementioned composition to amammal having a disorder that is responsive to LAG-3 inhibition orneutralization, whereupon the disorder is treated in the mammal. Adisorder that is “responsive to LAG-3 inhibition” or “responsive toLAG-3 neutralization” refers to any disease or disorder in which adecrease in LAG-3 levels or activity has a therapeutic benefit inmammals, preferably humans, or the improper expression (e.g.,overexpression) or increased activity of LAG-3 causes or contributes tothe pathological effects of the disease or disorder. Disorders that areresponsive to LAG-3 inhibition include, for example, cancer andinfectious diseases. The inventive method can be used to treat any typeof cancer known in the art, such as, for example, melanoma, renal cellcarcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer,colon cancer, gall bladder cancer, laryngeal cancer, liver cancer,thyroid cancer, stomach cancer, salivary gland cancer, prostate cancer,pancreatic cancer, or Merkel cell carcinoma (see, e.g., Bhatia et al.,Curr. Oncol. Rep., 13(6): 488-497 (2011)). The inventive method can beused to treat any type of infectious disease (i.e., a disease ordisorder caused by a bacterium, a virus, a fungus, or a parasite).Examples of infectious diseases that can be treated by the inventivemethod include, but are not limited to, diseases caused by a humanimmunodeficiency virus (HIV), a respiratory syncytial virus (RSV), aninfluenza virus, a dengue virus, a hepatitis B virus (HBV, or ahepatitis C virus (HCV)). Administration of a composition comprising theinventive immunoglobulin heavy chain polypeptide, the inventiveimmunoglobulin light chain polypeptide, the inventive LAG-3-bindingagent, the inventive nucleic acid sequence encoding any of theforegoing, or the inventive vector comprising the inventive nucleic acidsequence induces an immune response against a cancer or infectiousdisease in a mammal. An “immune response” can entail, for example,antibody production and/or the activation of immune effector cells(e.g., T-cells).

As used herein, the terms “treatment,” “treating,” and the like refer toobtaining a desired pharmacologic and/or physiologic effect. Preferably,the effect is therapeutic, i.e., the effect partially or completelycures a disease and/or adverse symptom attributable to the disease. Tothis end, the inventive method comprises administering a“therapeutically effective amount” of the LAG-3-binding agent. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve a desiredtherapeutic result. The therapeutically effective amount may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the LAG-3-binding agent to elicit adesired response in the individual. For example, a therapeuticallyeffective amount of a LAG-3-binding agent of the invention is an amountwhich decreases LAG-3 bioactivity in a human.

Alternatively, the pharmacologic and/or physiologic effect may beprophylactic, i.e., the effect completely or partially prevents adisease or symptom thereof. In this respect, the inventive methodcomprises administering a “prophylactically effective amount” of theLAG-3-binding agent. A “prophylactically effective amount” refers to anamount effective, at dosages and for periods of time necessary, toachieve a desired prophylactic result (e.g., prevention of diseaseonset).

A typical dose can be, for example, in the range of 1 μg/kg to 20 mg/kgof animal or human body weight; however, doses below or above thisexemplary range are within the scope of the invention. The dailyparenteral dose can be about 0.00001 μg/kg to about 20 mg/kg of totalbody weight (e.g., about 0.001 μg/kg, about 0.1 μg/kg, about 1 μg/kg,about 5 μg/kg, about 10 μg/kg, about 100 μg/kg, about 500 μg/kg, about 1mg/kg, about 5 mg/kg, about 10 mg/kg, or a range defined by any two ofthe foregoing values), preferably from about 0.1 μg/kg to about 10 mg/kgof total body weight (e.g., about 0.5 μg/kg, about 1 μg/kg, about 50μg/kg, about 150 μg/kg, about 300 μg/kg, about 750 μg/kg, about 1.5mg/kg, about 5 mg/kg, or a range defined by any two of the foregoingvalues), more preferably from about 1 μg/kg to 5 mg/kg of total bodyweight (e.g., about 3 μg/kg, about 15 μg/kg, about 75 μg/kg, about 300μg/kg, about 900 μg/kg, about 2 mg/kg, about 4 mg/kg, or a range definedby any two of the foregoing values), and even more preferably from about0.5 to 15 mg/kg body weight per day (e.g., about 1 mg/kg, about 2.5mg/kg, about 3 mg/kg, about 6 mg/kg, about 9 mg/kg, about 11 mg/kg,about 13 mg/kg, or a range defined by any two of the foregoing values).Therapeutic or prophylactic efficacy can be monitored by periodicassessment of treated patients. For repeated administrations overseveral days or longer, depending on the condition, the treatment can berepeated until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful and are within the scope ofthe invention. The desired dosage can be delivered by a single bolusadministration of the composition, by multiple bolus administrations ofthe composition, or by continuous infusion administration of thecomposition.

The composition comprising an effective amount of the inventiveimmunoglobulin heavy chain polypeptide, the inventive immunoglobulinlight chain polypeptide, the inventive LAG-3-binding agent, theinventive nucleic acid sequence encoding any of the foregoing, or theinventive vector comprising the inventive nucleic acid sequence can beadministered to a mammal using standard administration techniques,including oral, intravenous, intraperitoneal, subcutaneous, pulmonary,transdermal, intramuscular, intranasal, buccal, sublingual, orsuppository administration. The composition preferably is suitable forparenteral administration. The term “parenteral.” as used herein,includes intravenous, intramuscular, subcutaneous, rectal, vaginal, andintraperitoneal administration. More preferably, the composition isadministered to a mammal using peripheral systemic delivery byintravenous, intraperitoneal, or subcutaneous injection.

Once administered to a mammal (e.g., a cross-reactive human), thebiological activity of the inventive LAG-3-binding agent can be measuredby any suitable method known in the art. For example, the biologicalactivity can be assessed by determining the stability of a particularLAG-3-binding agent. In one embodiment of the invention, theLAG-3-binding agent (e.g., an antibody) has an in vivo half life betweenabout 30 minutes and 45 days (e.g., about 30 minutes, about 45 minutes,about 1 hour, about 2 hours, about 4 hours, about 6 hours, about 10hours, about 12 hours, about 1 day, about 5 days, about 10 days, about15 days, about 25 days, about 35 days, about 40 days, about 45 days, ora range defined by any two of the foregoing values). In anotherembodiment, the LAG-3-binding agent has an in vivo half life betweenabout 2 hours and 20 days (e.g., about 5 hours, about 10 hours, about 15hours, about 20 hours, about 2 days, about 3 days, about 7 days, about12 days, about 14 days, about 17 days, about 19 days, or a range definedby any two of the foregoing values). In another embodiment, theLAG-3-binding agent has an in vivo half life between about 10 days andabout 40 days (e.g., about 10 days, about 13 days, about 16 days, about18 days, about 20 days, about 23 days, about 26 days, about 29 days,about 30 days, about 33 days, about 37 days, about 38 days, about 39days, about 40 days, or a range defined by any two of the foregoingvalues).

The biological activity of a particular LAG-3-binding agent also can beassessed by determining its binding affinity to LAG-3 or an epitopethereof. The term “affinity” refers to the equilibrium constant for thereversible binding of two agents and is expressed as the dissociationconstant (K_(D)). Affinity of a binding agent to a ligand, such asaffinity of an antibody for an epitope, can be, for example, from about1 picomolar (pM) to about 100 micromolar (μM) (e.g., from about 1picomolar (pM) to about 1 nanomolar (nM), from about 1 nM to about 1micromolar (μM), or from about 1 μM to about 100 μM). In one embodiment,the LAG-3-binding agent can bind to an LAG-3protein with a K_(D) lessthan or equal to 1 nanomolar (e.g., 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.05 nM, 0.025 nM, 0.01 nM, 0.001nM, or a range defined by any two of the foregoing values). In anotherembodiment, the LAG-3-binding agent can bind to LAG-3 with a K_(D) lessthan or equal to 200 pM (e.g., 190 pM, 175 pM, 150 pM, 125 pM, 110 pM,100 pM, 90 pM, 80 pM, 75 pM, 60 pM, 50 pM, 40 pM, 30 pM, 25 pM, 20 pM,15 pM, 10 pM, 5 pM, 1 pM, or a range defined by any two of the foregoingvalues). Immunoglobulin affinity for an antigen or epitope of interestcan be measured using any art-recognized assay. Such methods include,for example, fluorescence activated cell sorting (FACS), separable beads(e.g., magnetic beads), surface plasmon resonance (SPR), solution phasecompetition (KINEXA™), antigen panning, and/or ELISA (see, e.g., Janewayet al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York,N.Y., 2001).

The LAG-3-binding agent of the invention may be administered alone or incombination with other drugs (e.g., as an adjuvant). For example, theLAG-3-binding agent can be administered in combination with other agentsfor the treatment or prevention of the diseases disclosed herein. Inthis respect, the LAG-3-binding agent can be used in combination with atleast one other anticancer agent including, for example, anychemotherapeutic agent known in the art, ionization radiation, smallmolecule anticancer agents, cancer vaccines, biological therapies (e.g.,other monoclonal antibodies, cancer-killing viruses, gene therapy, andadoptive T-cell transfer), and/or surgery. When the inventive methodtreats an infectious disease, the LAG-3-binding agent can beadministered in combination with at least one anti-bacterial agent or atleast one anti-viral agent. In this respect, the anti-bacterial agentcan be any suitable antibiotic known in the art. The anti-viral agentcan be any vaccine of any suitable type that specifically targets aparticular virus (e.g., live-attenuated vaccines, subunit vaccines,recombinant vector vaccines, and small molecule anti-viral therapies(e.g., viral replication inhibitors and nucleoside analogs).

In another embodiment, the inventive LAG-3 binding agent can beadministered in combination with other agents that inhibit immunecheckpoint pathways. For example, the inventive LAG-3 binding agent canbe administered in combination with agents that inhibit or antagonizethe programmed death 1 (PD-1), T-cell immunoglobulin domain and mucindomain 3 protein (TIM-3), and cytotoxic T-lymphocyte-associated protein4 (CTLA-4) pathways. Combination treatments that simultaneously targettwo or more of these immune checkpoint pathways have demonstratedimproved and potentially synergistic antitumor activity (see, e.g.,Sakuishi et al., J. Exp. Med., 207: 2187-2194 (2010); Ngiow et al.,Cancer Res., 71: 3540-3551 (2011); and Woo et al., Cancer Res., 72:917-927 (2012)). In one embodiment, the inventive LAG-3 binding agent isadministered in combination with an antibody that binds to TIM-3 and/oran antibody that binds to PD-1. In this respect, the inventive method oftreating a cancer or an infectious disease in a mammal can furthercomprise administering to the mammal a composition comprising (i) anantibody that binds to a TIM-3 protein and (ii) a pharmaceuticallyacceptable carrier or a composition comprising (i) an antibody thatbinds to a PD-1 protein and (ii) a pharmaceutically acceptable carrier.

In addition to therapeutic uses, the LAG-3-binding agent describedherein can be used in diagnostic or research applications. In thisrespect, the LAG-3-binding agent can be used in a method to diagnose adisorder or disease in which the improper expression (e.g.,overexpression) or increased activity of LAG-3 causes or contributes tothe pathological effects of the disease or disorder. In a similarmanner, the LAG-3-binding agent can be used in an assay to monitor LAG-3protein levels in a subject being tested for a disease or disorder thatis responsive to LAG-3 inhibition. Research applications include, forexample, methods that utilize the LAG-3-binding agent and a label todetect an LAG-3 protein in a sample. e.g., in a human body fluid or in acell or tissue extract. The LAG-3-binding agent can be used with orwithout modification, such as covalent or non-covalent labeling with adetectable moiety. For example, the detectable moiety can be aradioisotope (e.g., ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I), a fluorescent orchemiluminescent compound (e.g., fluorescein isothiocyanate, rhodamine,or luciferin), an enzyme (e.g., alkaline phosphatase,beta-galactosidase, or horseradish peroxidase), or prosthetic groups.Any method known in the art for separately conjugating anantigen-binding agent (e.g., an antibody) to a detectable moiety may beemployed in the context of the invention (see, e.g., Hunter et al.,Nature, 194: 495-496 (1962); David et al., Biochemistry, 13:1014-1021(1974); Pain et al., J. Immunol. Meth., 40: 219-230 (1981); andNygren, J. Histochem. and Cytochem., 30:407-412 (1982)).

LAG-3 protein levels can be measured using the inventive LAG-3-bindingagent by any suitable method known in the art. Such methods include, forexample, radioimmunoassay (RIA), and FACS. Normal or standard expressionvalues of LAG-3 can be established using any suitable technique, e.g.,by combining a sample comprising, or suspected of comprising, LAG-3 witha LAG-3-specific antibody under conditions suitable to form anantigen-antibody complex. The antibody is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound antibody. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, and radioactive materials (see, e.g., Zola, MonoclonalAntibodies: A Manual of Techniques, CRC Press, Inc. (1987)). The amountof LAG-3 polypeptide expressed in a sample is then compared with astandard value.

The LAG-3-binding agent can be provided in a kit, i.e., a packagedcombination of reagents in predetermined amounts with instructions forperforming a diagnostic assay. If the LAG-3-binding agent is labeledwith an enzyme, the kit desirably includes substrates and cofactorsrequired by the enzyme (e.g., a substrate precursor which provides adetectable chromophore or fluorophore). In addition, other additives maybe included in the kit, such as stabilizers, buffers (e.g., a blockingbuffer or lysis buffer), and the like. The relative amounts of thevarious reagents can be varied to provide for concentrations in solutionof the reagents which substantially optimize the sensitivity of theassay. The reagents may be provided as dry powders (typicallylyophilized), including excipients which on dissolution will provide areagent solution having the appropriate concentration.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example demonstrates a method of generating monoclonal antibodiesdirected against human LAG-3.

The gene encoding the extracellular domain (ECD) of human LAG-3 wasfused to either mouse IgG2a (human LAG-3 mIgG2a Fc) or a disabled formof wasabi fluorescent protein (dWFP human LAG-3) to produce antigen foruse in mouse immunization and hybridoma screening. Specifically, femaleSwiss Webster (SWR) mice were purchased from Harlan Laboratories, Inc.(Indianapolis, Ind.) and divided into two groups. After six days ofacclimatization, one group of animals was immunized with four to sixdoses of purified human LAG-3 mIgG2a Fc at 50 μg/mouse at intervals ofthree to four weeks using complete Freund's adjuvant (CFA) or incompleteFreunds adjuvant (IFA). The second group of animals was injected withfour to six doses at intervals of three to four weeks alternatingbetween human LAG-3 mIgG2a F or dWFP human LAG-3 ECD. CFA or IFA wasalso used as adjuvant in the second group. Animals were bled formeasurement of the serum titer to human LAG-3 as assessed by binding tocell surface human LAG-3. CHO-S cells were transfected with a fulllength human LAG-3 extracellular domain fused to the H-2Kk transmembranedomain (CHO-S huLAG-3 ECD cells). Sera were diluted from1:1,000-1:1,000,000 and incubated with the CHO-S huLAG-3 ECD cells for30 minutes at 4° C. Cells were centrifuged, washed once with PBS/1% BSA,and incubated with PE-conjugated (Southern Biotech, Birmingham, Ala.) orALEXAFLUOR™ 647- (Jackson Immunoresearch, West Grove, Pa.) labeled goatanti-mouse IgG (H+L) for 30 minutes at 4° C. Cells were washed twice inPBS/1% BSA, resuspended in PBS/I % BSA, and analyzed on a BD FACSARRAY™Bioanalyzer (BD Biosciences, Franklin Lakes, N.J.). Based on titerreadings, one animal from each group was boosted 3 days prior to spleencollection. Single cell suspensions were prepared from spleen tissue andused for generation of hybridomas by cell fusion using standardtechniques. Two different myeloma cell lines were used for fusion, FO(described in de St. Groth and Scheidegger, J. Immunol. Methods, 35:1-21 (1980)) and P3X63Ag8.653 (described in Kearney et al., J. Immunol.,123: 1548-1550 (1979)).

Hybridoma supernatants were screened for binding to CHO-S huLAG-3 ECDcells and compared to binding to untransfected CHO-S cells as describedabove. Based upon binding CHO-S huLAG-3 ECD cells, hybridomas weretransferred to 48-well plates and expanded.

Supematants were then tested for the ability to block binding of humanLAG-3 mIgG2a Fc labeled with DyL650 (human LAG-3 mIgG2a Fc DyL650) toDaudi cells, which is a B-cell line that endogenously expresses highlevels of MHCII (the LAG-3 receptor). Briefly, human LAG-3 mIgG2a FcDyL650 was pre-incubated with control IgG or anti-human LAG-3 candidatemonoclonal antibodies prior to addition to Daudi cells. Blocking wasmeasured by reduction in fluorescence to Daudi cells using a BDFACSARRAY™ Bioanalyzer. These hybridomas were then subcloned andexpanded to plate for generation of exhaust supernatant. Antibodies weresubsequently purified and retested to confirm both binding to CHO-ShuLAG-3 ECD cells and blocking ability in the Daudi assay.

The results of this example confirm the production of anti-LAG-3monoclonal antibodies using hybridoma cell technology.

Example 2

This example describes the design and generation of CDR-grafted andchimeric anti-LAG-3 monoclonal antibodies.

Antibodies from the hybridomas described in Example 1 were isotyped,subjected to RT-PCR for cloning the antibody heavy chain variable region(VH) and light chain variable region (VL), and sequenced. Specifically,RNA was isolated from cell pellets of hybridoma clones (1×10⁶cells/pellet) using the RNEASY™ kit (Qiagen, Venlo, Netherlands), andcDNA was prepared using oligo-dT-primed SUPERSCRIPT™ III First-StrandSynthesis System (Life Technologies, Carlsbad, Calif.). PCRamplification of the VL utilized a pool of degenerate mouse V_(L)forward primers (see Kontermann and Dubel, eds., Antibody Engineering,Springer-Verlag, Berlin (2001)) and a mouse a constant region reverseprimer. PCR amplification of the VH utilized a pool of degenerate mouseVH forward primers (Kontermann and Dubel, supra) and a mouse γ1 or γ2aconstant region reverse primer (based on isotyping of purified antibodyfrom each clone) with the protocol recommended in the SUPERSCRIPT™ IIIFirst-Stand Synthesis System (Life Technologies, Carlsbad, Calif.). PCRproducts were purified and cloned into pcDNA3.3-TOPO (Life Technologies,Carlsbad, Calif.).

Individual colonies from each cell pellet were selected and sequencedusing standard Sanger sequencing methodology (Genewiz, Inc., SouthPlainfield, N.J.). Variable region sequences were examined and alignedwith the closest human heavy chain or light chain V-region germlinesequence. Three antibodies were selected for CDR-grafting, which weredenoted (1) 5.B11, (2) 5.D7, and (3) 1.E10.

CDR-grafted antibody sequences were designed by cloning CDR residuesfrom each of the above-described mouse antibodies into the closest humangermline homolog. CDR-grafted antibody variable regions were synthesizedand expressed with human IgG/κ constant regions for analysis. Inaddition, mouse:human chimeric antibodies were constructed using thevariable regions of the above-described mouse antibodies linked to humanIgG/κ constant regions. Chimeric and CDR-grafted antibodies werecharacterized for binding to CHO-S huLAG-3 ECD cells and for activity inthe human LAG-3 ECD/Daudi blocking assay as described above.

The functional antagonist activity of chimeric and CDR-graftedantibodies also was tested in a human CD4⋅ T-cell:dendritic cell mixedlymphocyte reaction (MLR) assay in which activation of CD4⋅ T-cells inthe presence of anti-LAG-3 antibodies is assessed by measuring IL-2secretion. Because LAG-3 is a negative regulator of T-cell function,antagonism of LAG-3 was expected to result in increased T-cellactivation as measured by increased IL-2 production. The 5.B11, 5.D7,and 1.E10 CDR-grafted antibodies demonstrated antagonistic activity inthe MLR assay as measured by an increase in IL-2 activity.

The results of this example demonstrate a method of generating chimericand CDR-grafted monoclonal antibodies that specifically bind to andinhibit LAG-3.

Example 3

This example demonstrates affinity maturation of humanized monoclonalantibodies directed against human LAG-3.

CDR-grafted antibodies derived from two of the original murinemonoclonal antibodies described in Example 2, 5.D7 and 1.E10, weresubjected to affinity maturation via in silico somatic hypermutation(iSHM). This method incorporates mutations as predicted by computationalanalysis comparing in vivo matured antibody sequences, as downloadedfrom NCBI, and comparing them to germline human IGHV, IGKV, and IGLVsequences and their allelic forms (as described in Bowers et al., J.Biol. Chem., 288(11):7688-7696 (2013)). The LAG-3 binding properties ofresultant antibodies were assayed using surface plasmon resonance (SPR)as well as ability to bind to CHO-S huLAG-3 ECD cells as describedpreviously. Solution-based affinity analyses were also performed onusing a KINEXA™ 3000 assay (Sapidyne Instruments, Boise, Id.), andresults were analyzed using KINEXA™ Pro Software 3.2.6. Experimentalparameters were selected to reach a maximum signal with antibody alonebetween 0.8 and 1.2 V, while limiting nonspecific binding signal withbuffer alone to less than 10% of the maximum signal. Azlactone beads (50mg) were coated with antigen by diluting in a solution of human orcynoWFP-LAG-3 (50 μg/mL in 1 mL) in 50 mM Na₂CO₃. The solution wasrotated at room temperature for 2 hours, and beads were pelleted in apicofuge and washed twice with blocking solution (10 mg/mL BSA, 1 MTris-HCl, pH 8.0). Beads were resuspended in blocking solution (1 mL),rotated at room temperature for 1 hour, and diluted in 25 volumesPBS/0.02% NaN₃. For affinity measurement, the secondary antibody wasALEXFLUOR™ 647 dye-anti-human IgG (500 ng/mL). Sample antibodyconcentrations were held constant (50 pM or 75 pM), while human orcynomolgus WFP-LAG-3 antigen was titrated using a three-fold dilutionsseries from 1 μM to 17 pM. All samples were diluted in PBS, 0.2% NaN₃, 1mg/mL BSA and allowed to equilibrate at room temperature for 30 hours.Additionally, samples containing only antibody and only buffer weretested in order to determine maximum signal and nonspecific bindingsignal, respectively.

Thermal stability of the selected antibodies was assessed using aThermofluor assay as described in McConnell et al., Protein Eng. Des.Sel., 26: 151 (2013). This assay assesses stability through the abilityof a hydrophobic fluorescent dye to bind to hydrophobic patches on theprotein surface which are exposed as the protein unfolds. Thetemperature at which 50% of the protein unfolds (Tm) is determined tomeasure thermal stability. This assay demonstrated that 5.D7 monoclonalantibody variants had acceptable melting temperatures (Tms) (i.e.,greater than 70° C.) that were suitable for drug development.

De-risking of potential issues related to in vivo pharmokinetics of thetested antibodies was undertaken through assessment of non-specificbinding to target negative cells (see, e.g., Hotzel et al., mAbs, 4:753-760 (2012)). Antibodies were tested for binding to HEK 293f cellsusing a flow cytometry-based assay. The results indicated thatnon-specific binding was low for 5.D7 and could be further eliminatedthrough second step purification.

The results of this example confirm a method of affinity maturinghumanized monoclonal antibodies directed against LAG-3.

Example 4

This example demonstrates a method of identifying antibodies directedagainst human LAG-3 from an evolvable library.

An IgG evolvable library, based on germline sequence V-gene segmentsjoined to human donor-derived recombined (D)J regions, was constructedas described in Bowers et al. Proc. Natl. Acad Sci. USA, 108(51):20455-20460 (2011). IgG heavy chain (HC) and light chain (LC) werecloned into separate episomal vectors (Horlick et al., Gene, 243(1-2):187-194 (2000)), with each vector encoding a distinct antibioticselectable marker. The HC vector was formatted such that antibody waspresented both on the cell surface as well as secreted into the tissueculture medium (Horlick et al., J. Biol. Chem., 288(27): 19861-19869(2013)). The diverse sets of HCs and LCs were co-transfected into HEK293cells and expanded to approximately 10 cells. The cell library was thensubjected to two rounds each of negative selection against streptavidin(SA)-coupled magnetic beads alone (catalog #11047, Life Technologies,Carlsbad, Calif.) and irrelevant biotinylated antigen coated withSA-coupled magnetic beads. One round of positive selection was thenperformed using either magnetic beads coated directly with human LAG-3mIgG2a Fc or with SA-coupled magnetic beads coated with biotinylatedLAG-3 ECD mIgG1 Fc. The positively selected cells were diluted andplated in 96-well format at an approximate density of 1-10 cells/well.Resulting colonies were expanded into daughter plates and a portion ofeach population was tested for binding to LAG-3 ECD mIgG1 Fc DyL650 byFACSARRAY™ analysis. Antibodies secreted into the supernatant also weretested by BIACORE™ for ability to bind to LAG-3 ECD mIgG1 Fc.

Cells that showed specific staining to human LAG-3 mIgG2a Fc DyL650 byFACSARRAY™ analysis and/or binding by BIACORE™ were expanded for sortingand submitted for sequencing to recover the specific HC/LC combinationscapable of binding to human LAG-3. The open reading frames (ORFs)encoding the HCs and LCs of the antibodies found in the cell populationswere rescued by PCR. Generally, multiple HC/LC sequences were found bysequencing. In some cases the desired HC/LC combinations were identifiedby enriching cells expressing monoclonal antibodies of interest by firstFACS sorting with human LAG-3 mIgG2a Fc DyL650. Populations of cellsexhibiting high antibody expression and positive for binding to humanLAG-3 mIgG2a Fc DyL650 were isolated and subjected to subsequentsequence analysis. Overall, 12 different HC/LC pairs were identified aspotential specific anti-LAG-3 antibody hits suitable for furthercharacterization. These strategies were labeled A1/A14, A2, A3/A17,A4/A19, A5/A16, A6, A8/A20, A9, A10/A15, A11, A12, and A13.

Antibodies also were characterized for their ability to bind tocynomolgus monkey LAG-3 protein (cyno LAG-3). As these germlineantibodies identified from the library were too weak to bind to antigenexpressed on the cell surface, soluble antigen similar to the humanantigen was labeled with DyL650 (cyno LAG-3 mIgG2a Fc DyL650) and thenincubated with HEK293 cells displaying antibody strategies on the cellsurface. Eight antibody strategies identified from the evolvable librarywere tested and demonstrated an ability to bind to cyno LAG-3 ECD mIgG1Fc.

The results of this example confirm that monoclonal antibodies directedagainst human and non-human LAG-3 can be identified using an evolvablelibrary.

Example 5

This example demonstrates affinity maturation of antibodies directedagainst human LAG-3 identified using an evolvable library.

Stable cell lines co-expressing the HC and LC of each antibodyidentified from the evolvable library described in Example 4 weretransfected with activation induced cytidine deaminase (AID) to initiatein vitro SHM. AID was also transfected directly into the original mixedpopulation of cells expanded from the library screen. In all cases, cellpopulations were stained for both IgG expression and binding to antigen,collected by flow cytometry as a bulk population, and then expanded forsequence analysis by next generation sequencing (NGS). This process wasrepeated iteratively to accumulate SHM-derived mutations in the variableregions of both the heavy and light chains, and their derivatives, foreach strategy. Improvements in affinity were monitored by (1) SPR, (2)ability to bind to CHO-S huLAG-3 ECD cells, and (3) activity in the MLRassay. As the affinity of each antibody improved, the stringency ofselection was increased until affinity goals were achieved through theidentification and recombination of novel mutations.

Thermal stability of the selected antibodies was assessed using aThermofluor assay as described above. This assay demonstrated thatselect monoclonal antibodies from the A17 strategy had acceptable T_(m)sthat were suitable for drug development. Antibodies also were tested forbinding to HEK 293f cells using a flow cytometry-based assay. Theresults indicated that non-specific binding was low for select A17candidates.

Selected antibodies were tested for the ability to block binding ofhuman LAG-3 mIgG2a Fc labeled with DyL650 (human LAG-3 mIgG2a Fc DyL650)to Daudi cells, as described above. A dose range of neutralizingantibodies was preincubated with the soluble LAG-3 and analyzed by flowcytometry. Certain affinity-matured anti-LAG-3 antibodies completelyinhibited the interaction of soluble LAG-3 with MHCII.

The results of this example confirm a method of affinity maturingmonoclonal antibodies directed against LAG-3 identified using anevolvable library.

Example 6

This example demonstrates that an inventive anti-LAG-3 monoclonalantibody can inhibit LAG-3 signaling and enhance T-cell activation invitro alone and in combination with an anti-PD-1 antibody or ananti-TIM-3 antibody.

To establish parameters for anti-LAG-3 and anti-PD-1 combinationstudies, the anti-PD-1 antibody APE02058 was titrated in a dose-responsein the human CD4+ T-cell MLR assay described above. Based on the resultsfrom titrating the anti-PD-1 antibody in multiple MLR assays, 133 pM(approximate EC50) and 13 pM (approximate EC10) were selected fortesting in combination for antagonist studies with the anti-LAG-3monoclonal antibody. In combination with 133 pM or 13.3 pM of anti PD-1,the EC50 of the anti-LAG-3 monoclonal antibody decreased from 690 pM(anti-LAG-3 only) to 40 pM (+133 pM anti-PD-1) or 200 pM (+13.3 pManti-PD-1), which was a 17-fold and 3-fold increase in potency,respectively.

To establish parameters for anti-LAG-3 and anti-TIM-3 combinationstudies, the anti-LAG-3 antibody APE05505 was titrated in a doseresponse in the human CD4+ T-cell MLR assay described above. Based onthe results from titrating the anti-LAG-3 antibody in multiple MLRassays, 2 nM (approximate EC50) and 0.2 nM (approximate EC10) wereselected for testing in combination for antagonist studies with theanti-TIM-3 monoclonal antibody. In combination with 2 nM or 0.2 nM ofanti LAG-3, the EC50 of the anti-LAG-3 mAb decreased from 1InM(anti-LAG-3 only) to 6 nM (+0.2 nM anti-TIM-3) or 3 nM (+2 nManti-TIM-3), which was a 1.8-fold and 3.6-fold increase in potency,respectively.

The results of this example demonstrate that the inventive LAG-3 bindingagent can inhibit LAG-3 biological activity alone and in combinationwith antagonists of other negative regulators of the immune system.

Example 7

This example demonstrates that an inventive anti-LAG-3 monoclonalantibody can inhibit LAG-3 signaling and enhance T-cell activation invivo in combination with an anti-PD-1 antibody.

The activity of an anti-mouse LAG-3 surrogate monoclonal antibody (mAbC9B7W, BioXcell, West Lebanon, N.H.) was tested alone or in combinationwith an anti-mouse PD-1 surrogate monoclonal antibody (mAb RMP1-14,BioXcell, West Lebanon, N.H.) in the MC38 syngeneic tumor model. Groupsof ten animals were injected subcutaneously with 1×10⁶ MC38 cells. Tendays after inoculation, animals were randomized for tumor size. Micewere treated with 5 mg/kg of anti-PD-1 monoclonal antibody and/or 10mg/kg or anti-LAG-3 monoclonal antibody on days 1, 4, 8, and 11,totaling four doses of each antibody or combination of antibodies.Tumors were measured twice weekly to assess response to treatment. Theanti-PD-1+anti-LAG-3 combination was more efficacious in reducing tumorgrowth than each single agent alone. Complete response was observed inall ten animals of the group treated with the combination, as comparedto seven animals in the PD-1-only group and no animals in theanti-LAG-3-only group. Nine animals showing a complete response from thecombination group were then rechallenged by subcutaneous innoculationwith 4×10⁶ MC38 cells. None of the animals in the rechallenged groupdeveloped measurable tumor, while all control naive mice injected withthe same amount of cells grew palpable tumor.

The activities of the surrogate monoclonal antibodies described abovealso were tested alone or in combination in the Colon26 syngeneic tumormodel. Groups of 12 animals were injected subcutaneously with 5×10⁵Colon26 cells. Mice were treated with 10 mg/kg of anti-PD-1 antibodyand/or 10 mg/kg of anti-LAG-3 antibody on days 4, 7, 11, and 14,totaling four doses of each antibody or combination of antibodies.Tumors were measured twice weekly to assess response to treatment. Theanti-PD-1+anti-LAG-3 combination was more efficacious for tumor growththan each single agent alone. Complete response was observed in 10 outof 12 animals in the combination group, as compared to three animals inthe PD-1-only group and one animal in the anti-LAG-3-only group. Nineanimals showing complete response from the combination group were thenrechallenged with 5×10⁵ Colon26 cells. None of the animals in therechallenged group developed measurable tumor, while all the controlnaive mice injected with the same amount of cells grew palpable tumor.

The results of this example demonstrate that the inventive LAG-3 bindingagent, in combination with antagonists of other negative regulators ofthe immune system, can inhibit LAG-3 biological activity in vivo.

Example 8

This example demonstrates the effect of antibody isotype on anti-tumoractivity of an anti-LAG-3 antibody alone or in combination with ananti-PD-1 antibody in a syngeneic mouse tumor model.

Surrogate antibodies recognizing mouse LAG-3 of IgG1 (D265A) and IgG2aisotypes were created after sequencing and cloning the variable regionsof an anti-mouse LAG-3 neutralizing antibody (mAb C9B7W, BioXcell, WestLebanon, N.H.) from a rat hybridoma cell line and cloning into a mouseIgG1 or mouse IgG2a expression vector. These antibodies were then testedfor efficacy both alone and in combination with a mouse IgG (D265A)surrogate antibody recognizing mouse PD-1 similarly created from apurchased rat antibody from BioXcel (mAb RMP1-14, West Lebanon, N.H.).Specifically, Colon26 colon adenocarcinoma cells (5×10⁵ s.c.) wereimplanted into Balb/c mice and grown for 3 days. Mice were randomizedinto seven groups of 12 animals/group and dosed with each antibody orantibody combination on days 4, 7, 11, and 14 as set forth in Table 1.Mice injected with matched isotype antibodies served as a control. Tumorvolumes were measured twice weekly until the end of the study.

TABLE 1 Group Treatment Dose 1 Isotype IgG2a + Isotype IgG1(D265A) 10mg/kg, 1 mg/kg 2 Isotype IgG1 (D265A) 10 mg/kg 3 Anti-mPD-1 IgG1(D265A) 1 mg/kg 4 Anti-mLAG-3 IgG2a 10 mg/kg 5 Anti-mLAG-3 IgG1(D265A) 10 mg/kg6 Anti-mPD-1 IgG1(D265A) + 1 mg/kg, 10 mg/kg Anti-mLAG-3 IgG2a 7Anti-mPD-1 IgG1(D265A) + 1 mg/kg, 10 mg/kg Anti-mLAG-3 IgG1(D265A)

Results for this experiment are shown in FIGS. 1A and 1B, which showthat a single-agent anti-mouse LAG-3 antibody with minimal effectorfunction (i.e., IgG1 (D265A)) has anti-tumor efficacy as compared withan anti-mouse LAG-3 antibody with effector function (i.e., IgG2a), whichhas no apparent effect on tumor growth.

In addition, FIG. 1A shows an anti-mouse LAG-3 antibody with minimaleffector function (i.e., IgG1(D265A)) in combination with a regimen ofan anti-mouse PD-1 IgG1(D265A) antibody exhibited increased anti-tumoractivity compared with the anti-mouse PD-1 IgG1(D265A) antibody alone.However, an anti-mouse LAG-3 antibody with in-tact effector function(IgG2a) in combination with an anti-mouse PD-1 antibody was lessefficacious than anti-mouse PD-1 IgG1 (D265A) alone, suggesting that theeffector function of the antibody possibly interfered with anti-mousePD-1 mediated efficacy.

FIG. 1B provides graphs of tumor volume over time for individual animalsfrom treatment group 3 (anti-mouse PD-1 IgG1(D265A) antibody treatedanimals), group 7 (combination of anti-mouse PD-1 IgG(D265A) antibodywith anti-mouse LAG-3 IgG(D265A) antibody), and group 6 (combination ofanti-mouse PD-1 IgG1(D265A) antibody with anti-mouse LAG-3 IgG2antibody). In group 7 (anti-mouse PD-1 IgG1(D265A) antibody withanti-mouse LAG-3 IgG1(D265A)), 8/12 animals had no visible tumor growthby the end of the study. By contrast, only 3/12 animals in group 6(anti-mouse PD-1 IgG(D265A) antibody with anti-mouse LAG-3 IgG2antibody) had no visible tumor by the end of the study. In group 3(anti-mouse PD-1 IgG1 (D265A) alone), 6/12 animals were tumor free bythe end of study, suggesting possible interference by the effectorfunction of the anti-mouse LAG-3 IgG2 antibody when dosed in combinationwith the anti-mouse PD-1 IgG1 (D265A) antibody.

The results of this example demonstrate that anti-mouse LAG-3 andanti-mouse PD-1 antibodies without effector function, alone and incombination, can inhibit tumor growth in a mouse syngeneic tumor model.Efficacy was not observed using an anti-mouse LAG-3 antibody witheffector function and furthermore may interfere with anti-PD-1 mediatedefficacy.

Example 9

This example demonstrates that an inventive anti-LAG-3 monoclonalantibody inhibitory activity can be differentiated from that of ananti-PD-1 monoclonal antibody in a mixed lymphocyte reaction based upontime of harvest and correlates with PD-1 and LAG-3 expression.

A functional LAG-3 antagonist antibody was tested in a human CD4+ T-cellmixed lymphocyte reaction (MLR) assay in which activation of CD4+T-cells in the presence of anti-LAG-3 antibodies is assessed bymeasuring IL-2 secretion. The anti-LAG-3 antibody was tested side byside with an antagonistic anti-PD-1 antibody, wherein the antibodieswere added and/or harvested at different timepoints. Specifically,isolated peripheral blood monocytes from a human donor weredifferentiated into dendritic cells (DCs) and then mixed with CD4+T-cells isolated from a second donor. Inhibitory antibodies were addedeither at the start of the co-culture or 24 hours after the start of theco-culture. IL-2 levels were measured at 24 and 48 hours after antibodyaddition.

Antagonism of LAG-3 and PD-1 was expected to result in increased T-cellactivation as measured by increased IL-2 production. When added at thestart of the assay, the anti-PD-1 antibody increased IL-2 secretion atboth 24 and 48 hours post antibody addition, while the anti-LAG-3antibody increased IL-2 secretion when measured at 48 hours in the MLRassay, but not at 24 hours. When inhibitory anti-LAG-3 or anti-PD-1antibodies were added at 24 hours after starting the co-culture andharvested at 72 hours, both antibodies were active and the EC50 appearedto be equivalent (FIG. 2A). This correlates with expression as increasedPD-1 expression is observed at 24-72 hours, while LAG-3 appears to beexpressed later in the assay at 48 and 72 hours (FIG. 2B).

The results of this example demonstrate that the effects of LAG-3inhibition correlates with target expression, and that LAG-3 expressionoccurs temporally later than PD-1.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1-42. (canceled)
 43. A method of treating cancer in a human, said methodcomprising: administering to said human an effective amount of aLymphocyte Activation Gene-3 (LAG-3) binding antibody comprising: (i) animmunoglobulin light chain variable (VL) region comprising a light chainCDR1, a light chain CDR2, and a light chain CDR3 of SEQ ID NO:88; and,(ii) an immunoglobulin heavy chain variable (VH) region comprising aheavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of SEQ IDNO:
 182. 44. The method according to claim 43, wherein the cancer ismelanoma, renal cell carcinoma, lung cancer, bladder cancer, breastcancer, cervical cancer, colon cancer, gall bladder cancer, laryngealcancer, liver cancer, thyroid cancer, stomach cancer, salivary glandcancer, prostate cancer, pancreatic cancer, or Merkel cell carcinoma.45. The method according to claim 43, further comprising administeringan anti-PD-1 antibody to the human subject.
 46. The method according toclaim 43, further comprising administering an anti-TIM-3 antibody to thehuman subject.
 47. The method according to claim 43, wherein the LAG-3binding antibody is an IgG4 antibody.
 48. A method of treating cancer ina human, said method comprising: administering to said human aneffective amount of a Lymphocyte Activation Gene-3 (LAG-3) bindingantibody comprising: (i) an immunoglobulin light chain variable (VL)region comprising SEQ ID NO:88; and, (ii) an immunoglobulin heavy chainvariable (VH) region comprising SEQ ID NO:
 182. 49. The method accordingto claim 48, wherein the cancer is melanoma, renal cell carcinoma, lungcancer, bladder cancer, breast cancer, cervical cancer, colon cancer,gall bladder cancer, laryngeal cancer, liver cancer, thyroid cancer,stomach cancer, salivary gland cancer, prostate cancer, pancreaticcancer, or Merkel cell carcinoma.
 50. The method according to claim 48,further comprising administering an anti-PD-1 antibody to the human. 51.The method according to claim 48, further comprising administering ananti-TIM-3 antibody to the human.
 52. The method according to claim 48,wherein the LAG-3 binding antibody is an IgG4 antibody.
 53. A method oftreating lung cancer, colon cancer, or melanoma in a human, said methodcomprising: administering to said human an effective amount of aLymphocyte Activation Gene-3 (LAG-3) binding antibody comprising: (i) animmunoglobulin light chain variable (VL) region comprising a light chainCDR1, a light chain CDR2, and a light chain CDR3 of SEQ ID NO:88; and,(ii) an immunoglobulin heavy chain variable (VH) region comprising aheavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 of SEQ IDNO:
 182. 54. The method according to claim 53, further comprisingadministering an anti-PD-1 antibody to the human.
 55. The methodaccording to claim 53, further comprising administering an anti-TIM-3antibody to the human.
 56. The method according to claim 53, wherein theLAG-3 binding antibody is an IgG4 antibody.
 57. A method of treatinglung cancer, colon cancer, or melanoma in a human, said methodcomprising: administering to said human an effective amount of an IgG4Lymphocyte Activation Gene-3 (LAG-3) binding antibody comprising: (i) animmunoglobulin light chain variable (VL) region comprising SEQ ID NO:88;and, (ii) an immunoglobulin heavy chain variable (VH) region comprisingSEQ ID NO:
 182. 58. The method according to claim 57, further comprisingadministering an anti-PD-1 antibody to the human.
 59. The methodaccording to claim 57, further comprising administering an anti-TIM-3antibody to the human.